Human immunodeficiency virus (HIV) infection is the leading killer worldwide among infectious diseases, incurring 2-3 million deaths annually. Defining the mechanisms of HIV transmission and understanding the role of host cells that participate in the process are essential in developing effective strategies to combat HIV infection. Dendritic cells (DC) perform an important role in HIV infection and dissemination. DC-SIGN, a C- type lectin predominately expressed on myeloid DC, has been identified as a key mediator of DC-mediated HIV transmission. However, the precise mechanisms of DC-enhanced HIV infection and the role of DC-SIGN in the viral transmission process remain elusive. Our long-term objective is to elucidate the mechanisms of DC-mediated HIV dissemination and understand their role in HIV pathogenesis. The proposed studies will facilitate understanding of the mechanisms and the development of more effective interventions against HIV transmission, and potentially aid in development of novel HIV vaccine strategies. We hypothesize that cell- type-dependent HIV trafficking determines efficiency of DC-SIGN- or DC-mediated HIV transmission, and HIV Nef protein regulates DC-mediated HIV transmission through modulation of CD4 and DC-SIGN expression. This hypothesis is based on the observations that 1) DC-SIGN-mediated HIV transmission is cell-type dependent and requires cell-cell contact. The ability of DC-SIGN to promote HIV transfer correlates with the localization of the viral particles on the DC-SIGN-expressing cell; 2) CD4 coexpression in DC-SIGN transfectants abolishes HIV transmission to T cell targets. HIV Nef protein downregulates CD4 expression, but upregulates DC-SIGN expression and promotes HIV spread; 3) HIV transmission efficiency is significantly enhanced by maturation of DC, and the enhanced viral transmission is independent of DC- SIGN. Localization of HIV in immature DC is distinct from that in mature DC. The specific aims of this proposal are to: 1. Characterize cell-type restriction of DC-SIGN-mediated HIV transmission. 2. Examine the role of CD4 and Nef proteins in modulation of DC-SIGN-mediated HIV transmission. 3. Identify mechanisms of maturation of DC enhancing HIV transmission. These experimental designs will yield a better molecular and cellular description of HIV interactions with DC, which have relevance to control of HIV mucosal transmission and understanding of viral pathogenesis.